Use of n-beta-alanyl dopamine or a salt thereof for the preparation of a medicament for the prevention and treatment of liver fibrosis

Abstract

The application discloses application of N-beta-alanyldopamine or a salt thereof in preparation of a medicine for preventing and treating liver fibrosis, and belongs to the technical field of medicines. The application provides application of N-beta-alanyldopamine hydrochloride in preparation of a medicine for preventing and treating liver fibrosis. Experiments prove that, after N-beta-alanyldopamine hydrochloride is given, the content level of hydroxylysylpyridinoline (Pyr) is reduced, the hardness of a liver is reduced and the solubility of collagen is increased, thereby reducing collagen deposition in a liver tissue and relieving the symptom of liver fibrosis of a mouse induced by carbon tetrachloride (CCl4).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of N-β-alanyl dopamine or a salt thereof in the preparation of a drug for preventing and treating liver fibrosis. BACKGROUND

[0002] Liver fibrosis is a dynamic pathological process of all chronic liver diseases, which can be induced by hepatitis B or C virus, drugs, autoimmune and biliary diseases, alcoholic steatohepatitis and non-alcoholic steatohepatitis, etc. If not treated effectively, it can eventually lead to cirrhosis and related complications. The main feature of liver fibrosis is the excessive accumulation of extracellular matrix (ECM) maintained by heterogeneous liver myofibroblasts (MFs). The gradual deposition and remodeling of these ECM proteins in the lesion gap further leads to increased ECM hardness and disease progression. Among the complex components of ECM, collagen is the most important structural component, which, together with other components (elastic fibers, fibronectin, laminin, proteoglycans, etc.), provides a scaffold to support liver tissue structure and is the main determinant of tissue hardness.

[0003] ECM is a structural scaffold composed of non-cellular, fibrous and non-fibrous proteins, which can affect cell function and tissue homeostasis. Collagen is a large protein family that constitutes ECM and plays an important role in providing the structural and biomechanical integrity of tissues, and the cross-linking of collagen is also an important factor in preventing fibrosis from subsiding. The pattern and degree of lysyl hydroxylation of collagen affect the stability of collagen cross-linking, which provides tensile strength and mechanical stability of collagen fibrils. Two collagen cross-linking pathways have been identified, namely the lysine aldehyde (Lys ald )-mediated cross-linking pathway and the hydroxylysine aldehyde (Hyl ald )-mediated cross-linking pathway, and the cross-linking formed by Hyl ald is difficult to degrade. In fibrotic tissues, lysine hydroxylation of collagen is significantly increased, and the terminal peptide lysine residues are modified by procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2), and collagen cross-linking forms mature cross-linking products: hydroxylysylpyridinoline (Pyr). Our studies have confirmed that Plod2 expression is increased and Pyr content is increased in liver fibrosis, liver stiffness is increased and collagen solubility is decreased, and targeting Plod2 activity and function can improve liver fibrosis.

[0004] Although there are currently a number of drugs with anti-hepatic fibrosis effect into clinical research, but most of the toxic side effects, failed to achieve the clinical trial endpoint. So far, there has been no drug for liver fibrosis on the market. Therefore, the development of new target and mechanism of action of anti-hepatic fibrosis drugs have important value.

[0005] N-β-alanyldopamine (hydrochloride) is an organic compound, chemical formula is C 11 H 17 ClN2O3, is the main derivative of dopamine in blood, lymph. However, the prior art does not disclose any about N-β-alanyldopamine (hydrochloride) or its derivatives to relieve liver fibrosis. SUMMARY

[0006] The purpose of the present application is to provide N-β-alanyldopamine or its salt in the preparation of drugs for preventing and treating liver fibrosis, in order to solve the above problems existing in the prior art. The present application provides N-β-alanyldopamine hydrochloride (N-β-alanyldopamine (hydrochloride)) in the preparation of drugs for preventing and treating liver fibrosis, experiments show that, giving N-β-alanyldopamine hydrochloride reduces the content of hydroxylysylpyridine, reduces the hardness of liver and increases the solubility of collagen, and can relieve the symptoms of carbon tetrachloride (carbon tetrachloride, CCl4) induced liver fibrosis in mice.

[0007] To achieve the above purpose, the present application provides the following scheme:

[0008] The present application provides N-β-alanyldopamine or its salt in the preparation of drugs for preventing and treating liver fibrosis, the structural formula of the N-β-alanyldopamine is:

[0009] .

[0010] Optionally, the salt includes N-β-alanyldopamine hydrochloride.

[0011] Further, the structural formula of the N-β-alanyldopamine hydrochloride is:

[0012] .

[0013] Further, the N-β-alanyldopamine hydrochloride reduces the content of hydroxylysylpyridine in liver tissue, reduces the hardness of liver and increases the solubility of collagen, and further reduces the collagen deposition in liver tissue, thereby preventing and treating liver fibrosis.

[0014] Optionally, the dosage form of the drug includes an injection.

[0015] Optionally, the drug further comprises a pharmaceutically acceptable excipient.

[0016] The present application discloses the following technical effects:

[0017] The present application takes N-β-alanyldopamine (hydrochloride) as a representative compound, and evaluates the prevention and treatment effect of N-β-alanyldopamine (hydrochloride) on liver fibrosis through a mouse experiment. N-β-alanyldopamine (hydrochloride) is given to a mouse model of carbon tetrachloride-induced liver fibrosis through intraperitoneal injection within two consecutive weeks. The results show that N-β-alanyldopamine (hydrochloride) significantly reduces the content of hydroxylysylpyridinoline, reduces the hardness of the liver and increases the collagen solubility, and improves the pathological state of liver fibrosis.

[0018] The present application discloses the prevention and treatment effect and mechanism of N-β-alanyldopamine (hydrochloride) on liver fibrosis, and lays a theoretical foundation and technical support for the development of a new type of drug for liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0020] Figure 1 The figure shows the results of serological level detection and liver tissue section observation of mice after administration of different doses of N-β-alanyldopamine (hydrochloride). A is the result of alanine aminotransferase (ALT) level detection in the serum of mice in each dose group; B is the result of aspartate aminotransferase (AST) level detection in the serum of mice in each dose group; C is the liver tissue section observation figure of mice in each dose group;

[0021] Figure 2Figures 1A and 1B are photographs of representative pictures of Sirius red staining of mouse liver and statistical results of fibrosis area of mouse liver sections, respectively, for CCl4+Vehicle and CCl4+N-β-alanyldopamine (hydrochloride) groups;

[0022] Figure 3 Figure 2 is a statistical result of hydroxylysylpyridinoline (Pyr) content in mouse liver tissues for CCl4+Vehicle and CCl4+N-β-alanyldopamine (hydrochloride) groups;

[0023] Figure 4 Figures 3A and 3B are a schematic diagram of the detection process of mouse liver stiffness and statistical results of mouse liver stiffness, respectively, for CCl4+Vehicle and CCl4+N-β-alanyldopamine (hydrochloride) groups;

[0024] Figure 5 Figure 4 is a statistical result of collagen solubility in mouse liver for CCl4+Vehicle and CCl4+N-β-alanyldopamine (hydrochloride) groups. DETAILED DESCRIPTION

[0025] A number of exemplary embodiments of the present application are described herein, and those skilled in the art will understand that the present application is not limited to the embodiments described herein but can be practiced with modification and alteration within the scope and spirit of the present application. The description herein is therefore made by way of example only and is not intended to limit the scope of the present application.

[0026] It should be understood that the terms used herein are merely descriptive, but not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also encompassed within the scope of the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A description of the state of the art that can be made by a person of ordinary skill in the art, and further, that the reference is included for its

[0028] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the teachings of the description of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0029] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used to permit a possibility of additional features, components, steps, etc.

[0030] N-β-alanyldopamine: N-β-alanyl dopamine, CAS Registry Number 54653-62-2, has the following structural formula:

[0031] .

[0032] N-β-alanyldopamine (hydrochloride): N-β-alanyl dopamine hydrochloride, CAS Registry Number 58077-93-3, has the following structural formula:

[0033] .

[0034] Example 1

[0035] The experiments in this example were approved by the Ethics Committee of Capital Medical University. All experimental data are expressed as mean ± standard error, and the experimental results were analyzed using SPSS 25 statistical software. Independent sample t test was used for comparison between two groups, and one-way ANOVA method was used for comparison among multiple groups. Differences were considered significant at P < 0.05.

[0036] The animals used in the experiment were fed with ordinary diet and had free access to water. Five mice were housed in each cage in the SPF level animal room of the Capital Medical University Experimental Animal Center, which was maintained at (20 ± 2) °C with a 12-hour light / dark cycle. After one week of acclimatization, the mice were randomly divided into four groups and administered with four doses (0, 2.5 mg / kg body weight, 5 mg / kg body weight, and 10 mg / kg body weight) of N-β-alanyl dopamine (hydrochloride), with the 0 dose group of mice administered with an equal volume of PBS solution. Each group of mice was administered by intraperitoneal injection, twice a week for 2 weeks. The mice were sacrificed on day 14 for sampling, and were fasted for 6 hours before being sacrificed and given regular water.

[0037] ALT and AST levels in the serum of the mice were measured using ALT and AST detection kits (Nanjing Jiancheng, Nanjing, China), respectively. Before use, R1 and R2 solutions were prepared at a ratio of 200 μL:50 μL and preheated to 37°C. 10 μL of the test serum and a blank control (normal saline) were placed in a 96-well microtiter plate. 250 μL of the R1 and R2 mixture was added and mixed. The OD value was measured at a wavelength of 340 nm. The OD values ​​and the detection time points were recorded over a 6-minute period. Enzyme activity = (OD change per minute - OD change of blank) × 1746. Results are expressed as mean ± standard error. Experiments were repeated three times.

[0038] Test results such as Figure 1 A and Figure 1 As shown in Figure B, it can be seen that compared with normal mice (0 dose group), administration of 2.5 mg / kg body weight, 5 mg / kg body weight, and 10 mg / kg body weight of N-β-alanyldopamine (hydrochloride) had no significant effect on ALT and AST levels in the liver, indicating that N-β-alanyldopamine (hydrochloride) has no negative impact on liver function and has high biosafety.

[0039] The liver tissues of mice in each group were further taken for pathological sectioning and liver damage was assessed by hematoxylin-eosin (H&E) staining. The H&E staining steps were as follows:

[0040] (1) Conventional dewaxing of paraffin sections: xylene I for 10 minutes, xylene II for 10 minutes, 100% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes, 95% ethanol for 3-5 minutes, 90% ethanol for 3-5 minutes, 80% ethanol for 3-5 minutes, 70% ethanol for 3-5 minutes, 50% ethanol for 3-5 minutes, and distilled water for 2 minutes;

[0041] (2) H&E staining:

[0042] 1) Place the dewaxed sections in hematoxylin solution for 5 minutes;

[0043] 2) Place the slices in 0.5% hydrochloric acid alcohol for 5 seconds;

[0044] 3) Rinse the slices in tap water for 15 minutes and then in distilled water for a while;

[0045] 4) Stain the sections in eosin solution for 10 minutes;

[0046] (3) Rinse with tap water;

[0047] (4) Dehydration with gradient alcohol: 50% ethanol for 3-5 minutes, 70% ethanol for 3-5 minutes, 80% ethanol for 3-5 minutes, 90% ethanol for 3-5 minutes, 95% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes, and 100% ethanol for 3-5 minutes;

[0048] (5) Place the sections in xylene I for 10 minutes and xylene II for 10 minutes;

[0049] (6) Seal the slides with neutral gum to avoid air bubbles.

[0050] The results of pathological observation of mice in each group are as follows Figure 1 As shown in Figure C, the hepatocytes in all groups of mice exhibited regular morphology, uniform size, and neat arrangement. The hepatocyte nuclei were normal in morphology, with evenly distributed chromatin. There were no signs of nuclear division or abnormal nucleoli, and no obvious signs of inflammatory cell infiltration, fibrous tissue proliferation, or fibrosis. This indicates that administration of N-β-alanyldopamine (hydrochloride) at doses of 2.5 mg / kg, 5 mg / kg, and 10 mg / kg body weight has no toxic side effects on the normal liver and is highly safe.

[0051] Example 2

[0052] The experiments in this example were approved by the Ethics Committee of Capital Medical University. All experimental data are expressed as mean ± standard error. SPSS 25 statistical software was used to analyze the experimental results. The independent sample t test was used for comparison between two groups, and the one-way analysis of variance was used for comparison between multiple groups. P < 0.05 was considered to be a significant difference.

[0053] All animals used in the experiment were fed a standard diet and had free access to food and water. Mice were housed 5 per cage in an SPF-grade animal room at the Capital Medical University Experimental Animal Center at (20 ± 2)°C with a 12-hour light / dark cycle. After a week of pre-acclimatization, the mice were randomly divided into a model group and a treatment group.

[0054] The model group was induced with CCl4 to cause liver fibrosis in mice, and CCl4 [CCl4 / olive oil (OO)=1:9 (v / v)] was injected intraperitoneally at a dose of 1 mL / kg of body weight twice a week, and an equal volume of PBS solution was given 2 hours before CCl4 intraperitoneal injection, which was recorded as the CCl4+Vehicle group. The administration group was also induced with CCl4 to cause liver fibrosis in mice, and the administration method of CCl4 was the same as that of the model group; at the same time, N-β-alanyldopamine (hydrochloride) was injected intraperitoneally twice a week, and the administration time was 2 hours before CCl4 intraperitoneal injection, and the administration dose was 10 mg / kg of body weight, which was recorded as the CCl4+N-β-alanyldopamine (hydrochloride) group. On the 14th day of modeling, the mice were sacrificed for sampling, and the mice were fasted for 6 hours before being sacrificed, and the water was given regularly.

[0055] The liver tissues of mice in each group were taken for pathological section, and the fibrosis of liver tissues was evaluated by Sirius red staining method, and the Sirius red staining steps were as follows:

[0056] (1) Paraffin section routine deparaffinization: xylene I 10 minutes, xylene II 10 minutes, 100% ethanol 3-5 minutes, 100% ethanol 3-5 minutes, 95% ethanol 3-5 minutes, 90% ethanol 3-5 minutes, 80% ethanol 3-5 minutes, 70% ethanol 3-5 minutes, 50% ethanol 3-5 minutes, distilled water 2 minutes;

[0057] (2) The section was placed in 0.1% Sirius red staining solution, and stained at 37°C for 30 minutes;

[0058] (3) Tap water flow washing;

[0059] (4) Gradient alcohol dehydration: 50% ethanol 3-5 minutes, 70% ethanol 3-5 minutes, 80% ethanol 3-5 minutes, 90% ethanol 3-5 minutes, 95% ethanol 3-5 minutes, 100% ethanol 3-5 minutes, 100% ethanol 3-5 minutes;

[0060] (5) The section was placed in xylene I for 10 minutes and xylene II for 10 minutes;

[0061] (6) Neutral gum mounting, avoiding bubbles.

[0062] (7) Image analysis of liver tissue fibrosis:

[0063] The liver tissue sections were observed and photographed under an optical microscope. Ten different fields were randomly selected from each liver tissue section, and the fibrosis area was analyzed using ImageJ image analysis software. The total area S0 of each field was calculated, and then the total fibrous filament area S1 (red blood vessels were not included in S1) was circled. The blank area S2 (mainly the blank area of the blood vessel section) was then circled. The fibrosis quantitative value of the field was calculated according to the fibrosis area formula, and the average value of the fibrosis values of all fields of each section was calculated as the quantitative value of each section. The fibrosis area formula is as follows:

[0064]

[0065] The liver tissue section observation and statistical results of the two groups of mice are shown in Figure 2 It can be seen that the collagen fiber deposition in the liver of the CCl4+Vehicle group of mice is obvious, and the collagen fiber deposition in the CCl4+N-β-alanyldopamine (hydrochloride) group of mice is less. This indicates that after administration of the compound N-β-alanyldopamine (hydrochloride), the collagen fiber deposition in the mice is correspondingly reduced, indicating that N-β-alanyldopamine (hydrochloride) has a significant effect on liver fibrosis.

[0066] Example 3

[0067] The level of hydroxylysylpyridinoline (Pyr) in the liver tissue of the two groups of mice in Example 2 was detected using an ELISA kit, and the detection steps are as follows:

[0068] (1) The content of Pyr in the liver tissue was detected using a commercial ELISA kit (Huami Biotech, Wuhan, China) according to the manufacturer's instructions. First, the liver samples were ultrasonically treated (ice water bath, 30 minutes) according to the instructions;

[0069] (2) The various reagents were equilibrated at room temperature for at least 30 minutes, and the reagents were prepared according to the instructions and were ready for use;

[0070] (3) The enzyme-labeled plate was taken out, and one blank control well was set without any liquid: two wells were set for each standard point, and 50 μL of the corresponding standard was added to each well; the remaining each detection well was directly added with 50 μL of the sample to be tested;

[0071] (4) 50 μL of biotin-labeled material was added to each well (except for the blank control well), and mixed thoroughly. The non-dry adhesive seal was attached, and the plate was placed at 37°C for 1 hour;

[0072] (5) Wash the plate manually, discard the liquid in the well. Inject 200 μL / well of the washing solution into the well, stand for 10 seconds and spin dry. Repeat three times and then pat dry; wash the plate in the plate washer, select the three-time washing program, and pat dry after washing the plate;

[0073] (6) Add 50 μL of horseradish peroxidase-labeled avidin to each well (except for the blank control wells), mix thoroughly, seal with adhesive tape, and place at 37°C for 30 minutes;

[0074] (7) Wash the plate manually, discard the liquid in the well. Inject 200 μL into the well, stand for 10 seconds and spin dry. Repeat three times and then pat dry; wash the plate in the plate washer, select the three-time washing program, and pat dry after washing the plate;

[0075] (8) Add 50 μL of color developing agent A and 50 μL of color developing agent B to each well, mix well, and develop color at 37°C for 15 minutes in the dark. Add 50 μL of stop solution to each well;

[0076] (9) Measure the optical density (OD value) of each well in sequence at 450 nm using an enzyme marker.

[0077] The results of the detection of the level of pyridinoline (Pyr) in the liver tissues of the two groups of mice are shown in Table 1. Figure 3 As can be seen, compared with the CCl4+Vehicle group of mice, the level of Pyr per mole of collagen in the liver of the CCl4+N-β-alanyldopamine (hydrochloride) group of mice decreased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can reduce the level of Pyr in the liver and improve liver fibrosis.

[0078] Example 4

[0079] The hardness changes in the liver tissues of the two groups of mice in Example 2 were detected using an atomic force microscope, and the detection steps were as follows:

[0080] The liver frozen sections (5 μm thick) of the two groups of mice were immersed in a PBS solution containing protease inhibitors and placed on the operating table of the atomic force microscope. The MLCT probe (6 levers, 0.01-0.5 N / m) was lowered onto the tissue sample. The probe at the cantilever tip performed a two-dimensional scan of the sample in a 50×50 μm 2 region. For each indentation, the indentation speed was 20.3 μm / s, and the indentation depth was 50-80 nm. Three randomly selected regions were detected for each sample, and the hardness data were analyzed using NanoScopeAnalysis 1.9 software.

[0081] The results of the hardness detection in the liver tissues of the two groups of mice are shown in Table 2. Figure 4As shown in the figure, compared with the CCl4+Vehicle group of mice, the hardness of the liver of the CCl4+N-β-alanyldopamine (hydrochloride) group of mice decreased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can reduce the hardness of the liver and improve liver fibrosis.

[0082] Example 5

[0083] The changes in collagen solubility in the liver tissues of the two groups of mice in Example 2 were detected, and the detection steps were as follows:

[0084] (1) The liver tissue samples of the two groups of mice were placed in a neutral salt (Tris buffer salt solution containing protease inhibitors) solution and incubated on a 4°C rotary shaker overnight, centrifuged at 4°C at a speed of 14000 rpm for 30 minutes, and the supernatant was collected for collagen determination and defined as salt-soluble collagen;

[0085] (2) The precipitate was placed in a 0.5 mol / L acetic acid solution for extraction, incubated on a 4°C rotary shaker overnight, centrifuged at 4°C at a speed of 14000 rpm for 30 minutes, and the supernatant was collected for collagen determination and defined as acid-soluble collagen;

[0086] (3) The precipitate was placed in a 0.5 mol / L acetic acid solution containing 2 mg / mL pepsin, incubated on a 4°C rotary shaker overnight, centrifuged at 4°C at a speed of 14000 rpm for 30 minutes, and the supernatant was collected for collagen determination and defined as pepsin-soluble collagen. The precipitate was defined as insoluble collagen.

[0087] (4) The collagen content of each part was determined by hydroxyproline content determination method, and the total soluble collagen content was obtained by adding the contents of the three parts of soluble collagen, and statistical calculation was performed.

[0088] The results of collagen solubility in the liver tissues of the two groups of mice are shown in the figure Figure 5 As shown in the figure, compared with the CCl4+Vehicle group of mice, the insoluble collagen content in the liver of the CCl4+N-β-alanyldopamine (hydrochloride) group of mice decreased, and the soluble collagen content increased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can increase collagen solubility and improve liver fibrosis.

[0089] Therefore, the results of the present example show that N-β-alanyldopamine (hydrochloride) can play a role in treating liver fibrosis by reducing the content of Pyr, reducing the hardness of the liver, and increasing the solubility of collagen.

[0090] The above-described embodiments are merely preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art, without departing from the design spirit of the present application, should fall within the scope of the claims of the present application.

Claims

1. Use of N-β-alanyl dopamine or its salt in the preparation of a medicament for preventing and treating liver fibrosis, characterized in that: The structural formula of the N-β-alanyl dopamine is: 。 2. The use according to claim 1, characterized in that Such salts include N-β-alanyl dopamine hydrochloride.

3. The use according to claim 2, characterized in that The structural formula of the N-β-alanyl dopamine hydrochloride is: 。 4. The use according to claim 1, characterized in that The N-β-alanyl dopamine hydrochloride reduces the content of hydroxylysylpyridinoline in liver tissue, reduces liver hardness and increases collagen solubility, thereby reducing collagen deposition in liver tissue, thereby preventing and treating liver fibrosis.

5. The use according to claim 1, characterized in that The dosage form of the drug includes injection.

6. The use according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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